Antitumor Effects of Natural Bioactive Ursolic Acid in Embryonic Cancer Stem Cells.

Embryonic cancer cells (CSCs) could cause different types of cancer, a skill that makes them even more dangerous than other cancer cells. Identifying CSCs using natural products is a good option as it inhibits the recurrence of cancer with moderate various effects. Ursolic acid (UA) is a pentacyclic triterpenoid extracted from fruit and herbal remedies and has known anticancer functions against various cancer cells. However, its potential against CSCs remains uncertain. This study was planned to examine the induction of cell apoptosis by the UA. For cell signaling studies, we performed experiments, which are real-time qPCR and immunoblotting. Also, various cellular processes were analyzed using flow cytometry. The results raised a barrier to cell proliferation by the UA in NTERA-2 and NCCIT cells. Morphological studies also confirmed the UA's ability to cause cell death in embryonic CSCs. Examination of cell death importation showed that the UA formed the expression of the iNOS and thus the cell generation and mitochondrial reactive oxygen generation, which created a reaction to cellular DNA damage by raising the protein levels of phospho-histone ATR and ATM. In addition, the UA created the binding of the G0/G1 cell cycle to NTERA-2 and NCCIT cells, improved the expression levels of p21 and p27, and reduced the expression levels of CDK4, cyclin D1, and cyclin E, confirming the UA's ability to initiate cell cycle arrest. Finally, the UA created an internal mechanism of apoptosis in the embryonic CSC using BAX and cytochrome c regulation as well as the regulation of BCL-xL and BCL-2 proteins. Therefore, UA could be the best candidate for targeting CSCs and thus suppressing the emergence of cancer.

Non-toxic sulfur inhibits LPS-induced inflammation by regulating TLR-4 and JAK2/STAT3 through IL-6 signaling.

Janus kinase 2 (JAK2) and STAT3 signaling is considered a major pathway in lipopolysaccharide (LPS)­induced inflammation. Toll­like receptor 4 (TLR­4) is an inflammatory response receptor that activates JAK2 during inflammation. STAT3 is a transcription factor for the pro­inflammatory cytokine IL­6 in inflammation. Sulfur is an essential element in the amino acids and is required for growth and development. Non­toxic sulfur (NTS) can be used in livestock feeds as it lacks toxicity. The present study aimed to inhibit LPS­induced inflammation in C2C12 myoblasts using NTS by regulating TLR­4 and JAK2/STAT3 signaling via the modulation of IL­6. The 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide assay was conducted to analyze cell viability and reverse transcription polymerase chain reaction and western blotting performed to measure mRNA and protein expression levels. Chromatin immunoprecipitation and enzyme­linked immunosorbent assays were used to determine the binding activity of proteins. The results indicated that NTS demonstrated a protective effect against LPS­induced cell death and inhibited LPS­induced expression of TLR­4, JAK2, STAT3 and IL­6. In addition, NTS inhibited the expression of nuclear phosphorylated­STAT3 and its binding to the IL­6 promoter. Therefore, NTS may be a potential candidate drug for the treatment of inflammation.

Validation of exercise-response genes in skeletal muscle cells of Thoroughbred racing horses.

Objective: To understand the athletic characteristics of Thoroughbreds, high-throughput analysis has been conducted using horse muscle tissue. However, an in vitro system has been lacking for studying and validating genes from in silico data. The aim of this study is to validate genes from differentially expressed genes (DEGs) of our previous RNA-sequencing data in vitro. Also, we investigated the effects of exercise-induced stress including heat, oxidative, hypoxic and cortisol stress on horse skeletal muscle derived cells with the top six upregulated genes of DEGs. Methods: Enriched pathway analysis was conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool with upregulated genes in horse skeletal muscle tissue after exercise. Among the candidates, the top six genes were analysed through geneMANIA to investigate gene networks. Muscle cells derived from neonatal horse skeletal tissue were maintained and subjected to exercise-related stressors. Transcriptional changes in the top six genes followed by stressors were investigated using qRT-PCR. Results: The inflammation response pathway was the most commonly upregulated pathway after horse exercise. Under non-cytotoxic conditions of exercise-related stressors, the transcriptional response of the top six genes was different among types of stress. Oxidative stress yielded the most similar expression pattern to DEGs. Conclusion: Our results indicate that transcriptional change after horse exercise in skeletal muscle tissue strongly relates to stress response. qRT-PCR results showed that stressors contribute differently to the transcriptional regulation. These results would be valuable information to understand horse exercise in the stress aspect.

Effect of methylsulfonylmethane on oxidative stress and CYP3A93 expression in fetal horse liver cells.

OBJECTIVE: Stress-induced cytotoxicity caused by xenobiotics and endogenous metabolites induces the production of reactive oxygen species and often results in damage to cellular components such as DNA, proteins, and lipids. The cytochrome P450 (CYP) family of enzymes are most abundant in hepatocytes, where they play key roles in regulating cellular stress responses. We aimed to determine the effects of the antioxidant compound, methylsulfonylmethane (MSM), on oxidative stress response, and study the cytochrome P450 family 3 subfamily A (CYP3A) gene expression in fetal horse hepatocytes. METHODS: The expression of hepatocyte markers and CYP3A family genes (CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, and CYP3A97) were assessed in different organ tissues of the horse and fetal horse liver-derived cells (FHLCs) using quantitative reverse transcription polymerase chain reaction. To elucidate the antioxidant effects of MSM on FHLCs, cell viability, levels of oxidative markers, and gene expression of CYP3A were investigated in H2O2-induced oxidative stress in the presence and absence of MSM. RESULTS: FHLCs exhibited features of liver cells and simultaneously maintained the typical genetic characteristics of normal liver tissue; however, the expression profiles of some liver markers and CYP3A genes, except that of CYP3A93, were different. The expression of CYP3A93 specifically increased after the addition of H2O2 to the culture medium. MSM treatment reduced oxidative stress as well as the expression of CYP3A93 and heme oxygenase 1, an oxidative marker in FHLCs. CONCLUSION: MSM could reduce oxidative stress and hepatotoxicity in FHLCs by altering CYP3A93 expression and related signaling pathways.

Methylsulfonylmethane Induces Cell Cycle Arrest and Apoptosis, and Suppresses the Stemness Potential of HT-29 Cells.

BACKGROUND/AIM: Colorectal cancer is one of the most common malignancies worldwide. Small molecule-based chemotherapy is an attractive approach for the chemoprevention and treatment of colorectal cancer. Methylsulfonylmethane (MSM) is a natural organosulfur compound with anticancer properties, as revealed by studies on in vitro models of gingival, prostate, lung, hepatic, and breast cancer. However, the molecular mechanisms underlying the effects of MSM in colon cancer cells remain unclear. MATERIALS AND METHODS: Here, we investigated the effects of MSM, especially on the cell cycle arrest and apoptosis, in HT-29 cells. RESULTS: MSM suppressed the viability of HT-29 cells by inducing apoptosis and cell cycle arrest at the G0/G1 phase. MSM suppressed the sphere-forming ability and expression of stemness markers in HT-29 cells. CONCLUSION: MSM has anti-cancer effects on HT-29 cells, and induces cell cycle arrest and apoptosis, while suppressing the stemness potential.

Tannic Acid Inhibits Non-small Cell Lung Cancer (NSCLC) Stemness by Inducing G0/G1 Cell Cycle Arrest and Intrinsic Apoptosis.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is one among the most common cancers worldwide. Recently, dietary phytochemicals have been reported as an attractive approach to improve the symptoms of NSCLC patients. Tannic acid is a natural polyphenol, which is known to have anticancer effects on in vitro models of breast, gingival and colon cancer. However, the molecular mechanisms associated with the actions of tannic acid on A549 human lung cancer cells have not been elucidated. MATERIALS AND METHODS: In this study, we analyzed the effect of tannic acid on A549 cells and their underlying mechanisms using western blotting, flow cytometry, invasion assay and tumorsphere formation assay. RESULTS: Tannic acid treatment suppressed the viability of A549 cells through cell cycle arrest and induction of the intrinsic pathways of apoptosis. In addition, the various malignant phenotypes of A549 cells including invasion, migration, and stemness were inhibited by tannic acid treatment. CONCLUSION: Tannic acid could be used as an effective inhibitor of lung cancer progression.

Sulfur Compounds Inhibit High Glucose-Induced Inflammation by Regulating NF-κB Signaling in Human Monocytes.

High glucose-induced inflammation leads to atherosclerosis, which is considered a major cause of death in type 1 and type 2 diabetic patients. Nuclear factor-kappa B (NF-κB) plays a central role in high glucose-induced inflammation and is activated through toll-like receptors (TLRs) as well as canonical and protein kinase C-dependent (PKC) pathways. Non-toxic sulfur (NTS) and methylsulfonylmethane (MSM) are two sulfur-containing natural compounds that can induce anti-inflammation. Using Western blotting, real-time polymerase chain reaction, and flow cytometry, we found that high glucose-induced inflammation occurs through activation of TLRs. An effect of NTS and MSM on canonical and PKC-dependent NF-κB pathways was also demonstrated by western blotting. The effects of proinflammatory cytokines were investigated using a chromatin immunoprecipitation assay and enzyme-linked immunosorbent assay. Our results showed inhibition of the glucose-induced expression of TLR2 and TLR4 by NTS and MSM. These sulfur compounds also inhibited NF-κB activity through reactive oxygen species (ROS)-mediated canonical and PKC-dependent pathways. Finally, NTS and MSM inhibited the high glucose-induced expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and binding of NF-κB protein to the DNA of proinflammatory cytokines. Together, these results suggest that NTS and MSM may be potential drug candidates for anti-inflammation therapy.

Effect of Methylsulfonylmethane on Proliferation and Apoptosis of A549 Lung Cancer Cells Through G2/M Cell-cycle Arrest and Intrinsic Cell Death Pathway.

BACKGROUND/AIM: Methylsulfonylmethane (MSM) is a natural organic compound that displays anti-inflammatory as well as antioxidant properties. MSM reportedly has potential in inhibition of tumor cells. However, molecular mechanisms underlying the effects of MSM on lung cancer remain unclear. MATERIALS AND METHODS: In this study, the effect of MSM on A549 cells was examined. We focused on the mode of apoptosis induced by MSM and investigated alterations in the integrity of the outer membrane of mitochondria. RESULTS: Our results showed that MSM inhibited viability of A549 cells and changed the shape and permeability of nuclei. In addition, MSM induced G2/M arrest. MSM reduced the mitochondrial membrane potential and contributed to release of cytochrome c from mitochondria to cytoplasm. CONCLUSION: MSM is a potential anticancer agent for the treatment of lung cancer.

The Inhibitory Mechanisms of Tumor PD-L1 Expression by Natural Bioactive Gallic Acid in Non-Small-Cell Lung Cancer (NSCLC) Cells.

Non-small-cell lung cancer (NSCLC) is the most common lung cancer subtype and accounts for more than 80% of all lung cancer cases. Epidermal growth factor receptor (EGFR) phosphorylation by binding growth factors such as EGF activates downstream prooncogenic signaling pathways including KRAS-ERK, JAK-STAT, and PI3K-AKT. These pathways promote the tumor progression of NSCLC by inducing uncontrolled cell cycle, proliferation, migration, and programmed death-ligand 1 (PD-L1) expression. New cytotoxic drugs have facilitated considerable progress in NSCLC treatment, but side effects are still a significant cause of mortality. Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a phenolic natural compound, isolated from plant derivatives, that has been reported to show anticancer effects. We demonstrated the tumor-suppressive effect of GA, which induced the decrease of PD-L1 expression through binding to EGFR in NSCLC. This binding inhibited the phosphorylation of EGFR, subsequently inducing the inhibition of PI3K and AKT phosphorylation, which triggered the activation of p53. The p53-dependent upregulation of miR-34a induced PD-L1 downregulation. Further, we revealed the combination effect of GA and anti-PD-1 monoclonal antibody in an NSCLC-cell and peripheral blood mononuclear-cell coculture system. We propose a novel therapeutic application of GA for immunotherapy and chemotherapy in NSCLC.

A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA.

Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5' single-stranded tail in a 5' to 3' direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5'-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.

Non­toxic sulfur enhances growth hormone signaling through the JAK2/STAT5b/IGF­1 pathway in C2C12 cells.

Insulin­like growth factor­1 (IGF­1) regulates cell growth, glucose uptake and protein metabolism, and is required for growth hormone (GH) signaling­mediated insulin production and secretion. IGF1 expression is associated with STAT5, which binds to a region (TTCNNNGAA) of the gene. Although sulfur is used in various fields, the toxicity of this element is a significant disadvantage as it causes indigestion, vomiting, diarrhea, pain and migraine. Therefore, it is difficult to conduct in vitro experiments to directly determine the effects of dietary sulfur. Additionally, it is difficult to dissolve non­toxic sulfur (NTS). The present study aimed to identify the role of NTS in GH signaling as a Jak2/STAT5b/IGF­1 pathway regulator. MTT assay was used to identify an optimum NTS concentration for C2C12 mouse muscle cells. Western blotting, RT­PCR, chromatin immunoprecipitation, overexpression and small interfering RNA analyses were performed. NTS was dissolved in 1 mg/ml DMSO and could be used in vitro. Therefore, the present study determined whether NTS induced mouse muscle cell growth via GH signaling. NTS notably increased STAT5b binding to the Igf1 promoter. NTS also promoted GH signaling by upregulating GH receptor expression, similar to GH treatment. NTS enhanced GH signaling by regulating Jak2/STAT5b/IGF­1 signaling pathway factor expression in C2C12 mouse muscle cells. Thus, NTS may be used as a GH­enhancing growth stimulator.

Tannic Acid Promotes TRAIL-Induced Extrinsic Apoptosis by Regulating Mitochondrial ROS in Human Embryonic Carcinoma Cells.

: Human embryonic carcinoma (EC; NCCIT) cells have self-renewal ability and pluripotency. Cancer stem cell markers are highly expressed in NCCIT cells, imparting them with the pluripotent nature to differentiate into other cancer types, including breast cancer. As one of the main cancer stem cell pathways, Wnt/ß-catenin is also overexpressed in NCCIT cells. Thus, inhibition of these pathways defines the ability of a drug to target cancer stem cells. Tannic acid (TA) is a natural polyphenol present in foods, fruits, and vegetables that has anti-cancer activity. Through Western blotting and PCR, we demonstrate that TA inhibits cancer stem cell markers and the Wnt/ß-catenin signaling pathway in NCCIT cells and through a fluorescence-activated cell sorting analysis we demonstrated that TA induces sub-G1 cell cycle arrest and apoptosis. The mechanism underlying this is the induction of mitochondrial reactive oxygen species (ROS) (mROS), which then induce the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated extrinsic apoptosis pathway instead of intrinsic mitochondrial apoptosis pathway. Moreover, ribonucleic acid sequencing data with TA in NCCIT cells show an elevation in TRAIL-induced extrinsic apoptosis, which we confirm by Western blotting and real-time PCR. The induction of human TRAIL also proves that TA can induce extrinsic apoptosis in NCCIT cells by regulating mROS.

Methylsulfonylmethane inhibits cortisol-induced stress through p53-mediated SDHA/HPRT1 expression in racehorse skeletal muscle cells: A primary step against exercise stress.

Cortisol is a hormone involved in stress during exercise. The application of natural compounds is a new potential approach for controlling cortisol-induced stress. Tumour suppressor protein p53 is activated during cellular stress. Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are considered to be two of the most stable reference genes when measuring stress during exercise in horses. In the present study cells were considered to be in a 'stressed state' if the levels of these stable genes and the highly stress responsive gene p53 were altered. It was hypothesized that a natural organic sulphur-containing compound, methylsulfonylmethane (MSM), could inhibit cortisol-induced stress in racing horse skeletal muscle cells by regulating SDHA, HPRT1 and p53 expression. After assessing cell viability using MTT assays, 20 µg/ml cortisol and 50 mM MSM were applied to horse skeletal muscle cell cultures. Reverse transcription-quantitative PCR and western blot analysis demonstrated increases in SDHA, HPRT1 and p53 expression in cells in response to cortisol treatment, which was inhibited or normalized by MSM treatment. To determine the relationship between p53 and SDHA/HPRT1 expression at a transcriptional level, horse gene sequences of SDHA and HPRT1 were probed to identify novel binding sites for p53 in the gene promoters, which were confirmed using a chromatin immunoprecipitation assay. The relationship between p53 and SDHA/HPRT1 expression was confirmed using western blot analysis following the application of pifithrin-α, a p53 inhibitor. These results suggested that MSM is a potential candidate drug for the inhibition of cortisol-induced stress in racehorse skeletal muscle cells.

Silibinin inhibits in vitro ketosis by regulating HMGCS2 and NF-kB: elucidation of signaling molecule relationship under ketotic conditions.

Ketosis is a condition where ketone bodies are produced as an alternative energy source, due to insufficient glucose for energy production so that the body switches from carbohydrate metabolism to mostly fat metabolism. In this study, we examined the anti-ketosis effects of silibinin, a major active component of silymarin. We induced ketosis in FL83B mouse hepatocytes in vitro by culturing in low glucose media and compared results to hepatocytes maintained in high-glucose conditions. We quantified ß-hydroxybutyrate (BHB) levels with a colorimetric assay. In low-glucose conditions, silibinin reduced the amount of BHB produced, compared to high-glucose conditions; thus, silibinin exhibited an anti-ketotic effect. Ketone body formation during beta oxidation is mediated by 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) regulates the transcription of HMGCS2, and plays a vital role in BHB levels. We showed that silibinin inhibited the expression of HMGCS2 and NF-kB at transcriptional and translational levels. Silibinin also inhibited the nuclear translocation of NF-kB and its DNA binding activity. To elucidate the relationship between HMGCS2 and NF-kB, we tested inhibited and over-expressed NF-kB. We found that NF-kB acted as a positive regulator for HMGCS2 under ketosis treatment conditions.

Nobiletin Inhibits CD36-Dependent Tumor Angiogenesis, Migration, Invasion, and Sphere Formation Through the Cd36/Stat3/Nf-Κb Signaling Axis.

Targeted cancer therapy with natural compounds is more effective than nontargeted therapy. Nobiletin is a flavonoid derived from citrus peel that has anticancer activity. Cluster of differentiation 36 (CD36) is a member of the class B scavenger receptor family that is involved in importing fatty acids into cells. CD36 plays a role in tumor angiogenesis by binding to its ligand, thrombospondin-1 (TSP-1), and then interacting with transforming growth factor beta 1 (TGFß1). CD36 is implicated in tumor metastasis through its roles in fatty acid metabolism. This study investigated the molecular mechanisms underlying nobiletin's anticancer activity by characterizing its interactions with CD36 as the target molecule. We hypothesize that the anti-angiogenic activity of nobiletin involving its regulation of CD36 via signal transducer and activator of transcription 3 (STAT3) rather than through TSP-1. Gene analysis identified a Gamma interferon activation site (GAS) element in the CD36 gene promoter that acts as a STAT3 binding site, an interaction that was confirmed by ChIP assay. STAT3 interacts with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), suggesting that nobiletin also acts through the CD36/ (STAT3)/NF-κB signaling axis. Nobiletin inhibited CD36-dependent breast cancer cell migration and invasion as well as CD36-mediated tumor sphere formation. Taken together, these results suggest that nobiletin inhibits cancer stem cells in multiple ways.

Salidroside inhibits migration, invasion and angiogenesis of MDA­MB 231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2.

The major hallmarks of tumor progression are angiogenesis, migration and metastasis. Among the components of Rhodiola rosea, salidroside (p­hydroxyphenethyl-ß­d-glucoside) is one of the most potent, and is present in all Rhodiola species. Recent data have revealed the anticancer effects of salidroside; however, the mechanism underlying its ability to inhibit tumor angiogenesis remains unknown. The present study aimed to analyze how salidroside affects major factors involved in breast cancer, and to elucidate its ability to inhibit angiogenesis and invasion. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis and migration, which interacts with matrix metalloproteinases (MMPs). Specifically, MMPs act as a downstream target for STAT3. Using western blotting and reverse transcription-quantitative polymerase chain reaction analysis, the present study demonstrated that treatment of MDA­MB 231 triple-negative breast cancer (TNBC) cells with salidroside led to inhibition of invasion and migration markers, and of STAT3 signaling. Furthermore, in vitro angiogenesis analyses in human umbilical vein endothelial cells confirmed the anti-angiogenic activity of salidroside. An electrophoretic mobility shift assay also demonstrated that salidroside may inhibit the DNA-binding activity of STAT3, preventing STAT3 from binding to a novel binding site of the MMP2 gene promoter. In conclusion, the present results demonstrated that salidroside may downregulate the STAT3 signaling pathway, and inhibit cell viability, migration and invasion through MMPs in breast cancer cells.

Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER⁺ Breast Cancer Cells.

Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER⁺) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. We also investigated the migration and invasive ability of nobiletin in ER⁺ cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER⁺ breast cancer cells.

Methylsulfonylmethane Induces G1 Arrest and Mitochondrial Apoptosis in YD-38 Gingival Cancer Cells.

Gingival squamous cell carcinoma is a rare form of cancer that accounts for less than 10% of all head and neck cancers. Targeted therapies with natural compounds are of interest because they possess high efficacy with fewer side-effects. Methylsulfonylmethane (MSM) is an organic sulfur-containing compound with anticancer activities. The main goal of this study was to induce proliferation inhibition and apoptosis in the metastatic YD-38 cell line. MSM up-regulated expression of P21Waf1/Cip1 and P27Kip1 genes and down-regulated expression of cyclin D1 (CCND1) and CDK4. Moreover, treatment with MSM induced apoptosis and up-regulation of BAX in YD-38 cells. In accordance, the expression of the BCL-2 and BCL-XL, were inhibited, indicating the role of mitochondria in MSM-induced apoptosis. Analysis of mitochondrial integrity showed a loss of mitochondrial potential with an increased level of cytochrome c in the cytosol compared to mitochondria. Active CASPASE-3 (CASP3) was also observed, confirming that MSM-induced apoptosis is caspase-mediated.

Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.

Momilactone B Inhibits Ketosis In Vitro by Regulating the ANGPTL3-LPL Pathway and Inhibiting HMGCS2.

Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.